Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors

Abstract

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5'-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.

Fig. 1.. HESs for basal and substrate-coupled cycles differ by the conformation of the A-loops.

(A) Ribbon representation of the OF Pgp [Protein Data Bank (PDB) code 6C0V] with the N- and C-terminal halves colored with orange and cyan, respectively, and highlighting the positions of spin-label pairs as purple spheres. (B) Distance distributions in the TMDs and NBDs and (C and D) the A-loops obtained in nucleotide-free Pgp (Apo) and the HES (ADP-Vi) in the presence and absence of the substrate Ver in nanodiscs. The corresponding distributions predicted from the cryo-EM OF structure are shown as dashed lines. (D) Distance distributions for NBS2 in mixed micelles are shown for reference. Residue 92 is not resolved in the cryo-EM structure, precluding prediction of distance distributions.

Fig. 2.. Stimulation of ATP turnover by substrates entails stabilization of an asymmetric HES.

(A) Cytoplasmic view of the NBD dimer in the OF conformation in complex with ATP (red sticks) showing the A-loop (red spheres) spin-label pairs. (B) Distance distributions of the A-loop pairs highlighting the substrate dependence of the short-distance component (arrow) at NBS2. (C) Pgp inhibitors stabilize an HES with a distinct distance component (arrows) relative to substrates. (D) The asymmetric HES population for transport substrates is directly related to the activation energy of ATP turnover (Ln k_cat). Experiments in (B) to (D) were performed in nanodiscs. Distance distributions were obtained in the trapped (ADP-Vi) HES.

Fig. 3.. The high-affinity inhibitor zosuquidar stabilizes a distinct IF conformation of Pgp.

(A) Ribbon representation of zosuquidar-bound Pgp cryo-EM structure (PDB code 6FN1) highlighting the positions of spin-label pairs as purple spheres. (B) Distance distributions obtained in nanodiscs for apo-Pgp, zosuquidar-bound nucleotide-free Pgp, and the HES (ADP-Vi). Predicted distributions are shown as dotted lines. Arrows highlight components on the intracellular side of the TMD and at the A-loops that are either not observed or are minor components in apo-Pgp. The y axes in Figs. 1 and 3 are identical.

Fig. 4.. Model of Pgp transport and inhibition.

The basal cycle (middle panels, steps 1 and 2) entails conformational sampling by ATP-bound Pgp to enable NBD dimerization followed by population of a symmetric HES. The substrate-coupled cycle (steps 1, 3a, 4a, and 2) is initiated by substrate binding. As previously postulated (8, 16, 18), a transient conformation with one tightly bound ATP molecule is likely populated. Hydrolysis of one ATP stabilizes the asymmetric HES (step 4a), which consists of OO/OF conformations. Hydrolysis of the second ATP leads to the symmetric HES as in the basal cycle (step 2). Pgp inhibition (steps 1, 3b, and 4b) is initiated by stabilization of an IF conformation in which the extracellular sides of TMD is apo-like, whereas the intracellular sides of TMDs and NBDs are closer than the apo state. ATP hydrolysis proceeds through a heterogeneous HES. For each intermediate, we show cytoplasmic views of the NBDs highlighting the conserved A-loop tyrosines as purple spheres along with associated DEER distributions in nanodiscs.